GENE SILENCING MEDIATED BY PROMOTER HOMOLOGY OCCURS AT THE LEVEL OF TRANSCRIPTION AND RESULTS IN MEIOTICALLY HERITABLE ALTERATIONS IN METHYLATION AND GENE ACTIVITY

The promoter homology-dependent inactivation of a 35Spro-hygromycin ph osphotransferase (hpt) gene, which is present at the H-2 locus, by the multipurpose 271 silencing licus has been studied. The 271 locus can silence any gene under the control of the 35Spro as well as endogenous nitrite reductase (NiR) genes of tobacco because of the presence of a chimeric antisense gene (35Spro-RiN). All F-1 progeny of a cross betw een homozygous H-2 and 271 lines were sensitive to hygromycin and were chlorotic (a symptom of nitrogen deficiency). These phenotypes were a ccompanied by a reduction in the steady-state levels of Hyg and NIR tr anscripts. Transcriptional run-on experiments indicated, however, that while NiR silencing occurred post-transcriptionally, the hpt gene was inactivated at the transcriptional level; this was associated with in creased methylation of the 35Spro of the hpt gene. NiR gene expression recovered uniformly to wild-type levels in first generation backcross (BC1) progeny that did not inherit the 271 locus. In contrast, hygrom ycin resistance was only partially and non-uniformly regained among ad ult BC1 plants, Moreover, substantial silencing of the hpt gene could persist into the BC2 generation. Genomic sequencing demonstrated that the meiotic heritability of hpt silencing in the absence of the 271 lo cus was correlated with cytosine methylation primarily at CpG and CpNp G residues. Despite this residual methylation, H-2 loci weakened by an association with 271 did not acquire the ability to silence a 'naive' H-2 locus. Fluorescence in situ hybridization revealed that the 277 l ocus was located at a telomere. The results strengthen the distinction between silencing effects involving homology restricted to coding or promoter regions, respectively. The former is a posttranscriptional pr ocess that is meiotically reversible; the latter is due to transcripti onal inactivation and is associated with increased promoter methylatio n, which can lead to meiotically heritable reductions in target gene a ctivity. the relevance of these data for the melotic heritability of s ilencing, the non-transferability of silencing activity, and the basis of 271 silencing effects is discussed.